Design, synthesis, and evaluation of novel diphenyl ether derivatives against drug-susceptible and drug-resistant strains of?Mycobacterium tuberculosis

Article abstract of DOI:10.1111/cbdd.13379

In our efforts to develop druggable diphenyl ethers as potential antitubercular agents, a series of novel diphenyl ether derivatives (5a–f, 6a–f) were designed and synthesized. The representative compounds showed promising in vitro activity against drug-susceptible, isoniazid-resistant, and multidrug-resistant strains of Mycobacterium tuberculosis with MIC values of 1.56?μg/ml (6b), 6.25?μg/ml (6a–d), and 3.125?μg/ml (6b–c), respectively. All the synthesized compounds exhibited satisfactory safety profile (CC₅₀?>?300?μg/ml) against Vero and HepG2 cells. Reverse phase HPLC method was used to probe the physicochemical properties of the synthesized compounds. This series of compounds demonstrated comparatively low logP values. pKa values of representative compounds indicated that they were weak acids. Additionally, in vitro human liver microsomal stability assay confirmed that the synthesized compounds possessed acceptable stability under study conditions. The present study thus establishes compound 6b as the most promising antitubercular agent with acceptable drug-likeness.

Full text of DOI:10.1111/cbdd.13379

DR SIDHARTHA SANKAR KAR (Orcid ID : 0000-0002-5032-4770)
Article type : Research Article
Design, synthesis and evaluation of novel diphenyl ether derivatives against drug
susceptible and resistant strains of Mycobacterium tuberculosis
a
a
b
c
Sidhartha S. Kar , Varadaraj Bhat G. , Vishnu P. Shenoy , Indira Bairy , G. Gautham
a,
Shenoy *
a
Department of Pharmaceutical Chemistry, Manipal College of Pharmaceutical Sciences,
Manipal Academy of Higher Education, Manipal-576104, India.
b
Department of Microbiology, Kasturba Medical College, Manipal Academy of Higher
Education, Manipal-576104, India.
c
Melaka-Manipal Medical College, Manipal Academy of Higher Education, Manipal-576104,
India.
*
Corresponding author. Department of Pharmaceutical Chemistry, Manipal College of
Pharmaceutical Sciences, Manipal Academy of Higher Education, Manipal-576104, India.
Tel.:+91-820-2922482; fax: +91-820-2571998.
E-mail addresses: gautham.gs@manipal.edu (G Gautham Shenoy)
ABSTRACT
In our efforts to develop druggable diphenyl ethers as potential antitubercular agents,
a series of novel diphenyl ether derivatives (5a-f, 6a-f) were designed and synthesized. The
representative compounds showed promising in vitro activity against drug susceptible,
isoniazid resistant and multi drug resistant strains of Mycobacterium tuberculosis with MIC
values of 1.56 µg/mL (6b), 6.25 µg/mL (6a-d) and 3.125 µg/mL (6b-c) respectively. All the
synthesized compounds exhibited satisfactory safety profile (CC > 300 µg/mL) against
5
0
Vero and HepG2 cells. Reverse phase HPLC method was used to probe the physicochemical
properties of the synthesized compounds. This series of compounds demonstrated
comparatively low logP values. pKa values of representative compounds indicated that they
This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process, which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1111/cbdd.13379
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were weak acids. Additionally, in vitro human liver microsomal stability assay confirmed that
the synthesized compounds possessed acceptable stability under study conditions. The
present study thus establishes compound 6b as the most promising antitubercular agent with
acceptable drug-likeness.
KEY WORDS
Diphenyl ether; anti-tubercular agents; safety profile; physicochemical studies; human liver
microsomal stability.
Tuberculosis (TB) is a global pandemic and was responsible for the death of 1.3
million HIV-negative patients and 374,000 HIV positive people in the year 2016. Recent
WHO report has highlighted that TB ranks alongside HIV/AIDS as the world’s leading
infectious disease killer, while shining a spot light on progress made in several countries,
[
1]
including India. Campaign to reduce TB infections is seriously jeopardized by multi drug
resistant tuberculosis(MDR-TB), extremely drug resistant tuberculosis (XDR-TB) and total
[
2]
drug resistant (TDR) tuberculosis. There is an urgent need to develop safe and effective
drugs for the treatment of TB infection.
Mycobacterium tuberculosis (MTb) synthesizes fatty acid via the type II pathway,
which is absent in human beings. Enoyl-ACP Reductase (ENR) is a key regulatory enzyme of
the type II FAS pathway and it catalyzes the final step of type II fatty acid elongation cycle.
Inhibition of the fatty acid synthesis pathway is an attractive option since the drugs, which
inhibit this pathway, can be selectively toxic to MTb. Triclosan (1) (Figure 1), a diphenyl
ether derivative, targets mycobacterial ENR and exhibits MIC of 12.5 µg/ mL against
[3]
Mycobacterium tuberculosis H37Rv.
Unlike isoniazid (INH), triclosan does not require prior activation to bind with
[4]
MtbENR. From the literature reports on existing inhibitors of Mtb ENR, it can be proposed
that blocking the entry of natural substrate (acyl carrier protein) by the inhibitors like
triclosan, could be a plausible approach to disturb the essential reduction step in the
[
5]
mycobacterial mycolic acid biosynthesis. Thus, the attempts to find diphenyl ether based
[
6-9]
antiTB agents have geared up in the recent past.
Although triclosan carries the promise to
combat resistant strains of mycobacterium, its utility for human treatment is limited due to its
[
10]
poor solubility and sub-optimal bioavailability.
efficinet antitubercular agent is a major challenge.
Hence, transforming triclosan in to an
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[
11,12]
Encouraged with our previous experience with diphenyl ether scaffold
and
subsequent literature reports of antitubercular potential of related compounds, we decided to
carry out further studies and develop novel diphenyl ether derivatives as potential
antitubercular agents with improved druggability. Another purpose of this research work is to
get relevant information on the structural requirement of diphenyl ether scaffold to improve
its antitubercular potential.
1
1
. EXPERIMENTAL SECTION
.1. Methods and Materials
All moisture-sensitive reactions were carried out under nitrogen atmosphere in
anhydrous solvents. Column chromatography was carried out on 100-200 mesh silica gel.
Progress of the reactions was monitored by TLC using Aluminum backed sheets of silica gel
6
0 F24 (Merck). Melting points were recorded with a laboratory melting point apparatus and
1
13
are uncorrected. H NMR and C NMR spectra were recorded on a NMR Spectrometer
AV400-400 MHz High Resolution Multinuclear FT-NMR Spectrometer, Bruker) using
DMSO-d as the solvent. Mass spectrometry (MS) data were obtained on a LC-MS (Agilent
(
6
6
520 series, Q-TOF LC/MS) and GC-MS (Shimadzu GC-17A, GCMS-QP5050A). IR spectra
were recorded on Shimadzu Affinity-1 FT IR spectrophotometer. UV-Visible spectra were
recorded on Shimadzu UV-2450 spectrophotometer and λmax was recorded. The purity of
the final compounds was checked by reverse phase HPLC (UFLC, Shimadzu) using C-18
column in isocratic mode solvent systems (methanol and buffer, pH = 7.4) and found to be ≥
9
5%.
1
.1.1 Synthesis of 1-(3-methoxy-4-phenoxyphenyl) ethanone (2)
The 1-(3-methoxy-4-phenoxyphenyl) ethanone (2) was prepared as per the previously
[
12]
reported method
and purified by column chromatography over silica 100-200 with
hexane: ethyl acetate (8:2) as the mobile phase.
1
.1.2 Synthesis of 1-(3-hydroxy-4-phenoxyphenyl) ethanone (3)
The 1-(3-hydroxy-4-phenoxyphenyl) ethanone (3) was prepared as per the previously
[
12]
reported method
and purified by column chromatography over silica 100-200 with
hexane: ethyl acetate (6:4) as the mobile phase.
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1
1
.1.3 General method for synthesis of 1-(3-hydroxy-4-phenoxyphenyl)-3-aryl prop-2-en-
-one (4a-f)
The 1-(3-hydroxy-4-phenoxyphenyl)-3-aryl prop-2-en-1-one (4a-f) were prepared as
[
12]
per the previously reported method
and purified by column chromatography over silica
1
00-200 with hexane: ethyl acetate (6:4) as the mobile phase.
1
.1.4 General method for synthesis of 1-(3-hydroxy-4-phenoxyphenyl)-3-aryl propan-1-
one (5a-f)
To a solution of compounds 4a-f (2 mmol) in absolute alcohol (50 mL) and THF (10
mL) was added with 15 mL saturated aqueous solution of NH Cl (80 mmol) at 25-27 °C.
4
Then the reaction mixture was added with Zn (12 mmol) in four portions at 10 min. interval
and stirred for 6h at ambient temperature. Progress of the reaction was monitored by TLC,
using hexane: ethyl acetate (8:2) as the mobile phase. After the completion of reaction (6h),
the reaction mixture was filtered and the filtrate was evaporated under vacuum to remove the
volatiles. The residue obtained was added with ice cold water and extracted with ethyl acetate
(3x25 mL). The organic layers were separated, pooled, washed with water, brine, dried over
anhydrous MgSO and evaporated under vacuum. The crude compound was purified by
4
column chromatography over silica 100-200 with hexane: ethyl acetate (8:2) as the mobile
phase to afford the target compound.
1
.1.5 General method for synthesis of 5-(1-hydroxy-3-phenylpropyl)-2-phenoxyphenol
(6a-f)
To the solution of compounds 1-(3-hydroxy-4-phenoxyphenyl)-3-aryl propan-1-one
5a-f) (0.75 mmol) in methanol (4 mL) and THF (2 mL), NaBH (3.0 mmol) dissolved in aq.
(
4
NaOH (4N, 1 mL) was added in three portions and stirred at ambient temperature. Progress
of the reaction was monitored by TLC, using hexane: ethyl acetate (6:4) as the mobile phase.
After the completion of reaction (2h), the reaction mixture was quenched with saturated
NH Cl solution (5 mL) and evaporated under vacuum to remove the volatiles. The residue
4
obtained was added into ice cold water, neutralized to pH 7 (by adding dil. HCl) and
extracted with ethyl acetate (3x25 mL). The organic layers were separated, pooled, washed
with water, brine, dried over anhydrous MgSO and evaporated under vacuum. The crude
4
compound was purified by column chromatography over silica 100-200 with hexane: ethyl
acetate (6:4) as the mobile phase to afford the target compound.
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1
.2 In vitro antitubercular screening against drug susceptible strains of Mycobacterium
tuberculosis H37Rv and In vitro cell cytotoxicity screening
Procedure for MIC determination (micro plate alamar blue assay) against drug
[
12,13]
susceptible strains of Mycobacterium tuberculosis H37Rv has been reported previously.
Methods for the determination of CC (MTT assay) against Vero and HepG2 cells have
5
0
[
12,14,15]
been reported previously.
1
.3. In vitro antitubercular screening against drug resistant strains of Mycobacterium
tuberculosis
Selected compounds were screened against isoniazid resistant and multi drug resistant
MDR) strains of Mycobacterium tuberculosis H37Rv using mycobacterial growth indicator
tube (MGIT) assay method. Isoniazid resistant/ multi drug resistant Mycobacterium
(
[
16]
tuberculosis strain (clinical isolates) suspension was prepared as per the literature method.
It was diluted to afford a turbidity of 0.5 McFarland standard. 1 mL of the adjusted
suspension was diluted in 4 mL of sterile saline (1:5 dilution). About 0.1 mL of the isoniazid
resistant/multi drug resistant Mycobacterium tuberculosis (clinical isolates) strain suspension
was added into 10 mL of sterile saline to prepare the 1:100 growth control suspension and
mixed thoroughly. Test compounds were dissolved in DMSO and sterilized by filtering
through syringe filters (0.22 µm) to prepare stock solutions of concentration 10,000 µg/mL.
The stock solutions were diluted with saline water to afford working solutions of 80X
strength. Isoniazid and rifampicin were used as standard. Mycobacterium Growth Indicator
Tubes (BBL™ MGIT™, 7 mL, BD Bioscience, catalog No.245122), labeled with compound
codes and concentrations were added with 0.8 mL OADC, 0.1 mL test compounds and 0.5
mL of mycobacterial strain. Growth control tubes contained only OADC and Mycobacterial
culture. Each test sample was added to four MGITs at different concentrations and placed in a
tube holder with one growth control tube. Contents of tubes were mixed well. Then the tubes
were placed inside the BACTEC MGIT-900 instrument at 37 °C for 14 days. The BACTEC
MGIT-960 instrument monitored the fluorescence of the MGITs and the results were
reported. The results were analyzed by comparing the fluorescence of test sample tube and
growth control tube. The MIC was defined as the lowest concentration of drug that inhibited
bacterial growth.
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1
.4. Determination of logP, pKa and human liver microsomal stability assay
LogP, pKa and human liver microsomal stability of the compounds 5a-f and 6a-f were
[
12,17,18,19]
determined by previously reported methods.
2
2
. RESULTS AND DISCUSSION
.1 Design and chemistry
nd
Phenolic –OH at 2 position of the A-ring of triclosan (1, Fig. 1) plays a crucial role
in binding to the catalytic site of Mtb ENR. The presence of three chloro groups makes this
compound highly lipophilic and nonselective to InhA. Sivaraman et al. reported that ring-B
chloro substitutions in triclosan were involved in unfavorable steric interactions with the
[
7]
enzyme and their deletion from the scaffold increased the affinity 7-fold. In addition to this,
th
diphenyl ethers substituted with long alkyl chains at 4 position of ring-A have been reported
[
6]
to display better affinity towards Mtb ENR than triclosan. But those compounds were
highly lipophilic and showed low aqueous solubility. Hence design of structurally similar
th
diphenyl ether derivatives focused mainly on incorporating substituents at the 4 position and
nd
th
one –OH group at 2 position of ring-A. α, β- unsaturated ketone was introduced at the 4
position of ring-A of diphenyl ether moiety, forecasting that it would mimic the reactivity of
trans-2-enoyl acyl part of enoyl acyl carrier protein (Fig. 1) at the catalytic site of Mtb ENR.
Although the antitubercular activity of α, β- unsaturated ketones (chalcones) is already
reported, their carcinogenicity and metabolic instability make them unsuitable for lead
[20]
optimization. Thus the metabolically labile reactive conjugated system in the chalcones
was modified to lesser reactive corresponding non-conjugated ketones and alcohols.
Synthesis of this series of compounds is described in scheme-1. Acetovanillone was
taken as starting material and condensed with phenyl boronic acid by Chan-Lam O-arylation
reaction. Obtained ketone (compound 2) was subjected to BBr assisted demethylation
3
[21]
reaction as described by Gillmore et al. The Claisen-Schimidt condensation reaction of
compound 3 with different benzaldehydes afforded respective chalcones (compounds 4a-f).
Selective reduction of conjugated double bond of compounds 4a-f was optimized using
[
22]
Zn/NH Cl, where, Zn and NH Cl provided electron and proton.
Due to the weak
4
4
polarization, conjugated double bond of the chalcones was not affected by this mild reducing
agent and corresponding dihydro-chalcones 5a-f were obtained with 63.70% yield. In order to
increase the polar surface area, obtained dihydrochalcones (5a-f) were reduced further by
NaBH to give compounds 6a-f with 82.93% yield. Notably, NaBH assisted reduction of
4
4
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carbonyl group of compounds 5a-f to corresponding alcohols (6a-f) observed to be fastened
in presence of alkali (NaOH). Spectral data of all the synthesized compounds are included in
the supporting information.
2
.2. Biological evaluation
Compounds 5a-f, and 6a-f were screened against Mycobacterium tuberculosis
H37Rv. Their MIC results were given in Table 1. Safety profile (CC ) of this series of
5
0
compounds was established from MTT assay against Vero and HepG2 cells (Table 1).
Antitubercular potential of diphenyl ether derivatives was improved upon extension of
th
alkyl chain with bulky substituents at 4 position of ring A. Alkyl substituents of
dihydrochalcones bearing 4-F-Phenyl (5d) and furan ring (5f) demonstrated significant
antitubercular activity (MIC 10 µg/ mL). All dihydrochalcone derivatives (Compounds 5a-f)
showed acceptable safety profile against normal and human hepatoma cells (CC50 >300
µg/mL). Dramatic improvement in antiTB activity was observed when keto functionality of
dihydrochalcones (5a-f) was reduced to corresponding racemic alcohol derivatives (6a-f).
Interestingly, compounds 6a and 6c-f exhibited MIC of 4 µg/mL and compounds 6b and 6c
exhibited significant antitubercular activity with MIC of 1.56 µg/mL and 3.125 µg/mL
respectively. In this case, electron donating groups on phenyl substituent (6b-c) was observed
to be more favorable for antitubercular activity. A closer comparison between the MIC of
triclosan (MIC 12.5 µg/mL) and the compounds of the series, shows that, presence of
hydroxy substituent adjacent to ring A is critical for improving the antiTB activity. All the
compounds of this series achieved adequate safety profile (CC >300 µg/mL) against Vero
5
0
and HepG2 cells and demonstrated selective index greater than 10.
Most promising compounds 6a-f having MIC < 10 µg/mL against drug susceptible
strain of Mycobacterial tuberculosis H Rv were selected for antitubercular screening against
3
7
INH resistant and multi drug resistant (MDR) strains of Mtb (clinical isolates) (Table 2).
Compound 6b exhibited MIC of 6.125 µg/mL and 3.125 µg/mL against INH resistant and
multi drug resistant strains of Mycobacterium tuberculosis respectively. On the other hand,
compounds 6c and 6f retained their antitubercular potential against MDR strains.
Interestingly, compound 6e indicated antiTB activity at MIC of 4 µg/mL against both drug
susceptible and INH resistant strains of Mycobacterial tuberculosis.
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2
.3. Physicochemical studies
In order to probe the lipophilicity, experimental logP of synthesized was determined
by reverse phase HPLC method. In this method, the stationary phase simulated the biological
lipophilic membrane, and retention of the compounds was correlated with its lipophilicity.
Most of the synthesized compounds demonstrated moderate logP (Table 3). In-depth
analysis of logP of compounds indicated that there was no relationship between lipophilicity,
and observed antitubercular activity.
pKa of representative compounds was determined using Reverse phase HPLC
method. pKa was calculated from the retention factor (k') are shown in Table 3. Compound
5
b showed pKa 9.72. When the carbonyl group of 5b was replaced with an electron donating
secondary alcohol group in case of compounds 6b and 6e, the acidity of phenolic-OH was
shot up and resulted in pKa 6-6.24. With increased acidity, compounds 6b (MIC 1.56 µg/mL)
and 6e (MIC 4 µg/mL) showed substantial improvement in antitubercular activity. This may
be due to the fact that compounds having pKa near to 6 are capable of producing optimum
proportion of ionized and unionized species at biological pH (pH 7.4) to solubilize and
penetrate the membrane of the mycobacterium. This observation was totally in agreement
with the fact that, acidity of the drugs influenced their antitubercular activity.
Human liver microsome (HLM) stability (%) of selected compounds with promising
antitubercular activity was determined at different time points (1h and 2h). Presence of a
[10]
phenolic –OH group in triclosan moiety makes it susceptible for metabolism.
Hence,
phenolic –OH present in our diphenyl ether derivatives are theoretically susceptible to HLM
assisted metabolism. Nevertheless, in contrast to literature reports, HLM stability assay
results (Table 3) of our synthesized compounds indicated that they were stable when
incubated with human liver microsomes (retention > 50% after 1h).
CONCLUSION
In conclusion, novel diphenyl ether derivatives have been synthesized and evaluated
for their in vitro antitubercular activity. 6b was identified as the most promising compound
which exhibited MIC of 1.56 µg/mL, 6.25 µg/mL and 3.125 µg/mL respectively against drug
susceptible, isoniazid resistant and multi drug resistant (MDR) strains of Mycobacterium
tuberculosis. Safety profile of all synthesized compounds against Vero and HepG2 cells
(
CC >300µg/mL) was satisfactory. In the present study, we were able to decrease the
50
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intrinsic lipophilicity of diphenyl ether scaffold to an acceptable limit with improved
antitubercular potency against both drug susceptible and resistant strain. All the synthesized
diphenyl ether derivatives showed acceptable metabolic stability (retention > 50% after 1h)
against human liver microsomes. The present study indicates that there is further scope to
explore diphenyl ethers as antitubercular agents. To this end, work is in progress to do the
Mtb ENR enzyme assay of promising compounds and to elucidate the mechanism of action
on a molecular level.
ACKNOWLEDGEMENT
Research work described here is funded by Indian Council of Medical Research, New
Delhi. The authors are grateful to DST, New Delhi and Manipal Academy of Higher
Education, Manipal for providing necessary facilities to carry out the research work.
CONFLICT OF INTEREST
The authors declare no conflict of interest.
FIGURE 1 Design of compounds 5a-f and 6a-f.
SCHEME 1 Synthesis of compounds 5a-f and 6a-f.
1
Suppl. Fig. 1. H NMR (400 MHz, DMSO-d ) spectrum of compound 3
6
1
Suppl. Fig. 2. H NMR (400 MHz, DMSO-d ) spectrum of compound 3
6
1
Suppl. Fig. 3. H NMR (400 MHz, DMSO-d ) spectrum of compound 4a
6
1
Suppl. Fig. 4. H NMR (400 MHz, DMSO-d ) spectrum of compound 4b
6
1
Suppl. Fig. 5. H NMR (400 MHz, DMSO-d ) spectrum of compound 5a
6
1
Suppl. Fig. 6. H NMR (400 MHz, DMSO-d ) spectrum of compound 5b
6
1
Suppl. Fig. 7. H NMR (400 MHz, DMSO-d ) spectrum of compound 6a
6
1
Suppl. Fig. 8. H NMR (400 MHz, DMSO-d ) spectrum of compound 6b
6
1
3
Suppl. Fig. 9. C NMR (100 MHz, DMSO-d ) spectrum of compound 3
6
1
3
Suppl. Fig. 10. C NMR (100 MHz, DMSO-d ) spectrum of compound 4a
6
1
3
Suppl. Fig. 11. C NMR (100 MHz, DMSO-d ) spectrum of compound 4b
6
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1
3
Suppl. Fig. 12. C NMR (100 MHz, DMSO-d ) spectrum of compound 5a
6
1
3
Suppl. Fig. 13. C NMR (100 MHz, DMSO-d ) spectrum of compound 5b
6
1
3
Suppl. Fig. 14. C NMR (100 MHz, DMSO-d ) spectrum of compound 6a
6
1
3
Suppl. Fig. 15. C NMR (100 MHz, DMSO-d ) spectrum of compound 6b
6
Suppl. Fig. 16. ESI spectrum of compound 3
Suppl. Fig. 17. ESI spectrum of compound 4a
Suppl. Fig. 18. ESI spectrum of compound 4b
Suppl. Fig. 19. ESI spectrum of compound 5a
Suppl. Fig. 20. ESI spectrum of compound 5b
Suppl. Fig. 21. ESI spectrum of compound 6a
Suppl. Fig. 22. ESI spectrum of compound 6b
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